Leguminous plants, such as soybeans, are able to fix nitrogen from the atmosphere due to a symbiotic relationship between the plants and bacteria which dwell in nodules formed in the roots of the plants. Specifically, soil bacteria that are members of the family Rhizobiaceae, are capable of infecting plants and inducing highly differentiated root nodule structures within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant utilizes the ammonia as a source of nitrogen. The symbiotic root nodule bacteria are classified in several separate genera, including Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium.
Legume nodulation by rhizobia exhibits some species specificity. Bradyrhizobium species include the commercially important soybean nodulating strains B. japonicum (i.e., strains USDA 110 and 123), promiscuous rhizobia of the cowpea group, and B. parasponia (formerly Parasponia rhizobium) which nodulates the non-legume Parasponia, as well as a number of tropical legumes, including cowpea and siratro. The most important agricultural host of B. japonicum is soybean (Glycine max), but this bacterium will nodulate a few other legumes (e.g., cowpea and siratro). Fast growing rhizobia include, among others, Rhizobium etli, Sinorhizobium meliloti (formerly Rhizobium meliloti), and Rhizobium leguminosarum biovar trifolii, which nodulate bean, alfalfa, and clover, respectively. These Rhizobium species generally display a narrow host range. However, Rhizobium sp. NGR234 has the ability to nodulate over 100 genera of legumes. Sinorhizobium fredii (formerly Rhizobium fredii), is phylogenetically distinct from B. japonicum, but has the ability to nodulate Glycine soja (a wild soybean species), G. max cv. Peking, and a few other soybean cultivars.
There are currently about 70,000,000 acres of soybean grown in the United States. An inoculant industry exists to sell B. japonicum to farmers for incorporation into the soil during soybean planting. The use of these inoculants is intended to enhance the efficiency of nitrogen fixation. Unfortunately, for most of the United States, inoculation has been shown to be ineffective. Therefore, the inoculant industry remains relatively small (approximately $20-30 million per year). Indeed, at present, inoculation is only recommended for newly planted fields (i.e., those not planted with soybeans previously) and fields that have been out of production for over three years.
The primary reason for the inefficiency of soil inoculation is the presence of competing extant B. japonicum in soil. When a field has been producing soybean for more than one season, there is a build up of the B. japonicum populations in soil. These bacteria are highly competitive since they have adapted to their soil environment. Hence, when the inoculant is added, the indigenous soil B. japonicum strains compete and win the battle to nodulate the plant. The result is that, in many cases, less than 1% of the nodules formed on the planted soybean are due to the inoculant added. Therefore, even if a high-yielding B. japonicum strain is used as the inoculant, the farmer does not see the yield increase due to the fact that the inoculant has not found its way into the plant.
In the major soybean growing areas of the Midwest, the most competitive population of B. japonicum is that of serogroup 123. If improvement in the nitrogen fixing capacity of the soybean-Bradyrhizobium symbiosis through application of superior strains is to be realized, then the difficult problem of competition from indigenous populations (such as serogroup 123) will have to be solved.
Significant efforts have been made to understand and alter the competitiveness of indigenous Bradyrhizobia. For example, attempts to alter soybean nodule occupancy ratios of indigenous versus applied Bradyrhizobia have been reported. However, such alterations were only achieved by using ultra-high, economically infeasible rates of the applied strain. In a seven year study, Dunigan et al. [Agron. J. 76: 463-466 (1984)] demonstrated that the inoculant strain USDA 110 eventually formed the majority of nodules after high rates of application in the first 2 years (serogroup 123 was not among the indigenous population). However, the tenacious competitive ability of serogroup 123 appears not to be related to numbers per se and when normal rates of inoculant are applied the indigenous serogroup 123 population can still form up to 95% of the nodules on soybean.
The formation of nodules on leguminous plants involves a complex exchange and recognition of diffusible signals between the plant and the bacterial symbiont. A key plant signal are the flavonoids which trigger the induction of the bacterial nodulation genes (Day et al. [2000] In: Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process, ed. Triplett, E., Horizon Scientific Press, Norfolk, England, pp 385-414).
Nodulation genes of Bradyrhizobium and Rhizobium strains affect the early stages of nodule formation including host-bacterium recognition, infection and nodule development. Wild type strains of Bradyrhizobium species display some variation in these early nodulation steps which is reflected in differences in relative rates of initiation of nodulation and ultimately in differences in competitiveness between strains for nodule occupancy. For example, B. japonicum USDA 123 is believed to be more competitive for nodulation than B. japonicum USDA 110. Strains which initiate infection and nodules earlier will occupy a greater portion of the nodules on a given plant. Improving the competitiveness of a specific Bradyrhizobium is an important part of the development of improved inoculants for legumes. A more effective Bradyrhizobium strain must be able to out-compete the indigenous rhizobia population for nodule occupancy in order for their improved qualities to impact on the inoculated legume. Therefore, there is a significant need for an inoculating composition and/or an inoculating method which would improve competitiveness of a selected inoculant strain.
In the Bradyrhizobium japonicum-soybean symbiosis, several key regulatory components have been identified in the regulation of bacterial nodulation genes. Two of these, i.e., a LysR regulator, NodD1 and a two component regulatory system, NodWV are known to positively activate the B. japonicum nodulation genes in response to the plant produced isoflavonoids, genistein and daidzein. A third regulatory component (i.e., NolA) is a MerR type regulator (Sadowsky et al. [1991] Proc. Natl. Acad Sci. USA 88:637-641) that possesses the unique capacity to exist in three functionally distinct forms (i.e., NolA1, NolA2 and NolA3) (Loh et al. [1999] J. Bacteriol . 181:1544-1554). These polypeptides are derived from alternative translation of three in-frame initiation codons.
Induction of the B. japonicum nolA gene leads to the subsequent repression of the nodulation genes in this bacterium. The products of the nodulation genes are required for soybean nodulation. Thus, these plant compounds, by inducing nolA expression, lead eventually to an inhibition of nodulation.
NolA1 is required for the expression of both NolA2 and NolA3. Two transcriptional (P1 and P2) start sites have been identified (Loh et al. [1999] J. Bacteriol. 181:1544-1554). Transcription from P1 results in the formation of an mRNA encoding NolA1. NolA1 then regulates transcription from P2, resulting in the expression of both NolA2 and NolA3.
Although NolA is involved in the negative control of the nodulation genes (Dockendorff, T. C., J. Sanjuan, P. Grob, and G. Stacey [1994] Mol. Plant-Microbe Interact. 7:596-602), current information suggests that NolA does not act directly to repress nod gene expression. This view is supported by the observation that while expression of NolA from a multicopy plasmid resulted in a reduction of nod gene expression, interposon mutations to the nolA gene did not lead to elevated levels of nod gene expression (Garcia, M. L., J. Dunlap, J. Loh, and G. Stacey [1996] Mol. Plant-Microbe Interact 9:625-635). In fact, NolA appears to positively regulate the expression of NodD2, the latter of which has been shown to be a repressor of the nod genes in Rhizobium spp. NGR234, Bradyrhizobium spp. (Arachis) NC92 and Bradyrhizobium japonicum (Garcia, M. L., J. Dunlap, J. Loh, and G. Stacey [1996] Mol. Plant-Microbe Interact 9:625-635; Gillette, W. K. and G. H. Elkan [1996] J. Bacteriol . 178:2757-2766; and Fellay, R., M. Hanin, G. Montorzi, J. Frey, C. Freiberg, W. Golinowski et al. [1998] Mol. Microbiol. 27:1039-1050. Therefore, NolA affects repression indirectly, through the control of nodD2 expression.
Cell-cell signaling plays a large role in the ability of bacteria to respond and adapt to a particular environment. Regulatory systems that control gene expression in response to population density (i.e., quorum sensing) govern such bacterial phenotypes as bioluminescence, antibiotic production, plasmid conjugal transfer and the synthesis of virulence factors in both plant and animal pathogens (Hardman, A. M. et al. [ 1998] Antonie van Leeuwenhoek 74:199-210). Quorum sensing involves the recognition of self-produced signal compounds, which function to regulate the expression of genes when threshold levels of these signals have accumulated in cultures of a sufficiently high population density. hi Gram-negative bacteria, the best studied of these signals are N-Acyl homoserine-lactones (AHL) (Fuqua, W. C. et al. [1994] J. Bacteriol 176:269-275). In Gram-positive bacteria, an equivalent role is played by various posttranslationally-modified peptides (Kleerebezem, M. et al. [1997] Mol. Microbiol. 24:895-904). Several AHL compounds have been identified from rhizobia, including Rhizobium leguminosarum biovars viciae, trifoli and phaseoli, Rhizobium etli, and Rhizobium meliloti (Thorne and Williams [1999] J. Bacteriol. 181:981-990; Cha et al. [1998] Mol. Plant Microbe Int. 11:1119-1129; Gray et al. [1996] J. Bacterial. 178:372-376; Rosemeyer et al. [1998] J. Bacteriol. 180:815-821; VanBrussel et al. [1985] J. Bacteriol. 162:1079-1082; and Wijffelman et al. [1983] Mol. Gen. Genet. 192:171-176). In a few cases, these autoinducers have been implicated in the nodulation process. For example, the small AHL molecule produced by R. leguminosarum by. viciae is required for the expression of the rhiABC operon, which is important for rhizosphere growth and nodulation efficiency (Cubo et al. [1992] J. Bacteriol. 174:4026-4035). In R. etli, mutations that disrupt AHL synthesis resulted in decreased nodule numbers on host plants (Rosemeyer et al. [1998] J. Bacteriol. 180:815-821). Therefore, AHL-mediated quorum sensing may play an important role in the symbiotic process. To date, no quorum-sensing compound has been identified from the soybean symbiont Bradyrhizobium japonicum. 
The current invention addresses the inefficiency of soil inoculation due to the presence of competing indigenous B. japonicum in soil and provides novel compounds and compositions which increase the efficiency of nodulation in target plants. Specifically, field inoculants comprising high-yielding NolA insensitive B. japonicum and nolA inducers address the long standing obstacle of inefficient nodulation due to indigenous B. japonicum strains.